Molecular cloning and identification of cDNA recombinants coding for bovine prochymosin in E. coli.

Poly(A) RNA was isolated from the gastric mucosa of the bovine fourth stomach (the abomasum) using and analysing several calves not older than 12 days. The amount of the preprochymosin mRNA in the mucosa of those animals at best reaches about 5-10% of the poly(A) RNA as estimated by in vitro translation and immunoprecipitation. Starting from that material double-stranded complementary DNA was synthesized, inserted by dG dC tailing into the PstI site of the vector plasmid pBR322 and used for transformation of E. coli. Tetracycline resistant clones containing DNA sequences coding for the full length of prochymosin were recognized by colony hybridization with five specific d-oligonucleotides corresponding either to the N-terminal, the middle or the C-terminal part of prochymosin. Six recombinants were detected by screening of 1 500 recombinants with an oligonucleotide which corresponds to positions 649 to 663 of the nucleotide sequence published by Harris et al. (1982). Two of them were found to cover together the complete prochymosin sequence as evidenced by both positive colony hybridization with either the N-terminal or the C-terminal oligonucleotide probe, as well as by the restriction pattern of the selected plasmids.